Pediatric brain tumors are the third most frequent malignancy of children, and brain tumors are the leading cause of death in children with cancer. Medulloblastoma is the most common pediatric tumor of the cerebellum. These tumors can seed along the neuraxis and metastasize to extraneural tissue. During brain development, embryonic neuroepithelial cells migrate outwards and laterally to form the external granular layer of the cerebellum. Primitive neuroectodermal tumors (PNET), like medulloblastomas and basal cell carcinomas (BCC) of the skin, have been associated with two inherited cancer syndromes: Gorlin's and Turcot's. Gorlin's Syndrome, also called nevoid basal cell carcinoma syndrome (NBCCS), is an autosomal-dominant disease characterized by a range of tumor types such as BCC, medulloblastoma, ovarian fibroma, meningioma, fibrosarcoma, rhabdomyosarcoma and cardiac fibroma. Three percent of patients with Gorlin's develop medulloblastoma and Turcot patients are 92-fold more likely to develop medulloblastoma than the general population.
There is accumulating evidence that medulloblastomas result from the molecular dysregulation of the hedgehog (Hh) pathway, in particular sonic hedgehog (SHh), smoothen (Smo), patched (Ptch) and the transcription factor family, Gli1-3. The hh family of genes and their control in mammalian embryonic development is certainly pivotal. SHh plays a number of significant roles in embryonic development including the development of the cerebellum. SHh is produced by Purkinje cells and by granule neuron progenitor cells and is a mitogenic factor for granule neurons as well as a differentiation factor for Bergmann glial cells. In fact, cerebellum hyperproliferation appears to be the result of increased levels of SHh and its prolonged expression. Indeed, SHh has been shown to be associated with medulloblastomas from studies involving transgenic mice that over express SHh and in a transgenic human tissue model.
It is also known that mutations in ptch are responsible for Gorlin's Syndrome. In these individuals, one copy of the ptch gene is mutated, resulting in many of the heterozygous cases of medulloblastoma. Between 12 and 40% of non-inherited BCC arise from inactivation of both alleles of ptch. Ptch mutations, along with several other members of the Hh signaling pathway, also have been directly implicated in the development of medulloblastomas.
Cubis interruptus (Ci) is the terminal component of the Hh pathway, mediating transcriptional activation of hh target genes in response to Hh. Binding sites for Ci have been identified upstream to the promoters of both wg and ptch. There is a high degree of sequence homology between ci and the vertebrate Gli family of transcription factors. In vertebrates there are three homologs of the ci gene: gli1, gli2 and gli3, each having its own distinct pattern of expression. The Gli proteins are large transcription factors that bind DNA in a sequence specific manner via the last three fingers of their five zinc-finger domain. Gli1 is the most potent activator. Gli2 & 3 are thought to have dual functions both as a modified full-length activator and as a truncated processed repressor. Gli1 is constitutively activated in BCC, NBCCS and medulloblastoma.
Since its discovery in plants, post-transcriptional gene silencing has become an important tool in molecular biology. It was shown early on that gene silencing was mediated through a diffusable trans-acting product and later that this trans-acting factor was double-stranded RNA (dsRNA). Both antisense and sense RNA were able to shut down expression of a target gene. Gene silencing studies have shown that dsRNA are more effective at suppressing target genes than anti-sense or sense-strands alone. Only a few molecules of dsRNA are required to attain complete gene silencing. This dsRNA effect has been termed RNA interference (RNAi). RNAi can also be induced by transfecting cells with plasmids that express siRNAs. Furthermore, plasmids containing a sequence encoding a hairpin-forming, 45-50mer double-stranded RNA molecule termed small hairpin RNA (shRNA) under the control of an RNA Polymerase III (Pol 111) promoter when transfected into mammalian cells, have been shown to be more stably expressed, more efficient at reducing the levels of both exogenous and endogenous gene products and provide longer term reduction in target gene than siRNAs alone.
Many researchers are now using RNAi as a tool to ascertain the function of genes because it allows one to create ‘loss-of-function’ phenotypes quickly and easily. RNAi may also hold promise as a gene-specific therapeutic for the treatment of infectious diseases and cancer.